LC-MS/MS based metabolomics reveals the mechanism of skeletal muscle regeneration

Skeletal muscle possesses a robust regenerative capacity and can effectively repair itself following injury. However, research on the metabolic changes during skeletal muscle regeneration in large animals remains relatively limited. Therefore, in this study, we used pigs as a model and applied non-targeted LC-MS/MS metabolomic technology to reveal the metabolic changes during skeletal muscle regeneration, and conducted an in-depth exploration of important signaling pathways, which can provide a reference for further research on the mechanisms promoting skeletal muscle regeneration.

In this study, we used 18 piglets aged 35 days and weighing 7.10 ± 0.90 kg to construct a skeletal muscle regeneration model. These piglets were randomly divided into three treatment groups (n = 6) and injected with cardiotoxins (CTX) in the right longissimus dorsi muscle. They were euthanized on the 1st, 4th, and 16th days post-injection to collect right longissimus dorsi muscle samples as the treatment group. Additionally, the left longissimus dorsi muscle of piglets on the 4th day post-injection was selected as the control group. Phenotypic changes in skeletal muscle regeneration were determined through H&E staining, immunofluorescence, and Western Blot analysis, and LC-MS/MS untargeted metabolomics technology was utilized to explore the differential expressed metabolites (DEMs) involved in skeletal muscle regeneration.

Phenotyping results showed that the regeneration model showed 3 stages of inflammation, regeneration and remodeling, which indicated successful model construction. Non-targeted LC-MS/MS metabolomics analysis showed significant differences in the structure of metabolites in these 3 stages. (1) In the inflammatory stage, a total of 198 DEMs were identified, which were mainly enriched in the pathways regulating the inflammatory response. (2) in the repair stage, 264 DEMs were identified, which were mainly enriched in pathways that inhibit inflammatory response and promote protein synthesis. (3) During the remodeling stage, 102 DEMs were identified, which were mainly enriched in the pathways that inhibit protein depletion and promote protein deposition. Temporal expression analysis revealed metabolites consistent with changes in the skeletal muscle regeneration process and found that these metabolite functions were mainly enriched in inhibiting inflammatory responses, alleviating myofibrillar lysis, and promoting muscle growth. Among them, (R)-Lipoic acid, 8-Hydroxyguanosine, and Uridine 5'-monophosphate maybe key metabolites associated with skeletal muscle regeneration.

The skeletal muscle regeneration mechanism was systematically explored, and the metabolite time series analysis during skeletal muscle regeneration revealed some key metabolites that reflect the degree of skeletal muscle damage.